Twelve facultatively anaerobic, endophytic diazotrophs were isolated from surface-sterilized roots of the wild rice species and characterized by phenotypic and molecular methods. Six isolates were grouped together as group A by phenotypic characters, and this grouping was confirmed by SDS-PAGE whole-cell protein patterns and insertion sequence-based PCR (IS-PCR) methods. Phylogenetic analysis of the 16S rRNA gene sequence indicated that group A, represented by strain Ola 51, is closely related to D5/23 (98.9 % similarity, except that D5/23 has a 70 bp insertion) and (98.0 % similarity to the type strain). gene sequence analysis also showed strain Ola 51 has the highest sequence similarity to DSM 16656 (98.3 %), but supported the distinct position. Biological and biochemical tests, protein patterns, genomic DNA fingerprinting, antibiotic resistance and comparison of cellular fatty acids showed differences among group A, DSM 16656 and ATCC 13047. DNA–DNA hybridization distinguished strain Ola 51 from closely phylogenetically related species. Based on these data, the novel species sp. nov. is proposed, with strain Ola 51 (=LMG 24251 =CGMCC 1.7012) as the type strain.


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  1. Baldani, V. L. D. & Doebereiner, J.(1980). Host-plant specificity in the infection of cereals with Azospirillum spp. Soil Biol Biochem 12, 433–439.[CrossRef] [Google Scholar]
  2. Brenner, D. J., McWhorter, A. C., Kai, A., Steigerwalt, A. G. & Farmer, J. J., III(1986).Enterobacter asburiae sp. nov., a new species found in clinical specimens, and reassignment of Erwinia dissolvens and Erwinia nimipressuralis to the genus Enterobacter as Enterobacter dissolvens comb. nov. and Enterobacter nimipressuralis comb. nov. J Clin Microbiol 23, 1114–1120. [Google Scholar]
  3. Chaintreuil, C., Giraud, E., Prin, Y., Lorquin, J., Ba, A., Gillis, M., de Lajudie, P. & Dreyfus, B.(2000). Photosynthetic bradyrhizobia are natural endophytes of the African wild rice Oryza breviligulata. Appl Environ Microbiol 66, 5437–5447.[CrossRef] [Google Scholar]
  4. Chen, W. X., Yan, G. H. & Li, J. L.(1988). Numerical taxonomy study of fast-growing soybean rhizobia and proposal that Rhizobium fredii be assigned to Sinorhizobium gen. nov. Int J Syst Bacteriol 38, 392–397.[CrossRef] [Google Scholar]
  5. Cole, R. M. & Popkin, T. J.(1981). Electron microscopy. In Manual of Methods for General Bacteriology, pp. 34–51. Edited by P. Gerhardt, R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, N. R. Krieg & G. H. Phillips. Washington, DC: American Society for Microbiology.
  6. De Ley, J., Cattoir, H. & Reynaerts, A.(1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133–142.[CrossRef] [Google Scholar]
  7. Drancourt, M., Bollet, C., Carta, A. & Rousselier, P.(2001). Phylogenetic analyses of Klebsiella species delineate Klebsiella and Raoultella gen. nov., with description of Raoultella ornithinolytica comb. nov., Raoultella terrigena comb. nov. and Raoultella planticola comb. nov. Int J Syst Evol Microbiol 51, 925–932.[CrossRef] [Google Scholar]
  8. Eckert, B., Weber, O. B., Kirchhof, G., Halbritter, A., Stoffels, M. & Hartmann, A.(2001).Azospirillum doebereinerae sp. nov., a nitrogen-fixing bacterium associated with the C4-grass Miscanthus. Int J Syst Evol Microbiol 51, 17–26. [Google Scholar]
  9. Elbeltagy, A., Nishioka, K., Sato, T., Suzuki, H., Ye, B., Hamada, T., Isawa, T., Mitsui, H. & Minamisawa, K.(2001). Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species. Appl Environ Microbiol 67, 5285–5293.[CrossRef] [Google Scholar]
  10. Engelhard, M., Hurek, T. & Reinhold-Hurek, B.(2000). Preferential occurrence of diazotrophic endophytes, Azoarcus spp., in wild rice species and land races of Oryza sativa in comparison with modern races. Environ Microbiol 2, 131–141.[CrossRef] [Google Scholar]
  11. Farmer, J. J., III, Davis, B. R., Hickman-Brenner, F. W., McWhorter, A., Huntley-Carter, G. P., Asbury, M. A., Riddle, C., Wathen-Grady, H. G., Elias, C. & Fanning, G. R.(1985). Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. J Clin Microbiol 21, 46–76. [Google Scholar]
  12. Fujie, T., Huang, Y. D., Higashitani, A., Nishimura, Y., Iyama, S., Hirota, Y., Yoneyama, Y. & Dixon, R. A.(1987). Effect of inoculation with Klebsiella oxytoca and Enterobacter cloacae on dinitrogen fixation by rice-bacteria associations. Plant Soil 103, 221–226.[CrossRef] [Google Scholar]
  13. Gao, J. L., Sun, J. G., Li, Y., Wang, E. T. & Chen, W. X.(1994). Numerical taxonomy and DNA relatedness of tropical rhizobia isolated from Hainan province of China. Int J Syst Bacteriol 44, 151–158.[CrossRef] [Google Scholar]
  14. Hoffmann, H., Stindl, S., Ludwig, W., Stumpf, A., Mehlen, A., Heesemann, J., Monget, D., Schleifer, K. H. & Roggenkamp, A.(2005a). Reassignment of Enterobacter dissolvens to Enterobacter cloacae as E. cloacae subspecies dissolvens comb. nov. and emended description of Enterobacter asburiae and Enterobacter kobei. Syst Appl Microbiol 28, 196–205.[CrossRef] [Google Scholar]
  15. Hoffmann, H., Stindl, S., Stumpf, A., Mehlen, A., Monget, D., Heesemann, J., Schleifer, K. H. & Roggenkamp, A.(2005b). Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance. Syst Appl Microbiol 28, 206–212.[CrossRef] [Google Scholar]
  16. Hurek, T., Handley, L. L., Reinhold-Hurek, B. & Piche, Y.(2002).Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Microbe Interact 15, 233–242.[CrossRef] [Google Scholar]
  17. Iversen, C., Mullane, N., McCardell, B., Tall, B. D., Lehner, A., Fanning, S., Stephan, R. & Joosten, H.(2008).Cronobacter gen. nov., a new genus to accommodate the biogroups of Enterobacter sakazakii, and proposal of Cronobacter sakazakii gen. nov., comb. nov., Cronobacter malonaticus sp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov., Cronobacter genomospecies 1, and of three subspecies, Cronobacter dublinensis subsp. dublinensis subsp. nov., Cronobacter dublinensis subsp. lausannensis subsp. nov. and Cronobacter dublinensis subsp. lactaridi subsp. nov. Int J Syst Evol Microbiol 58, 1442–1447.[CrossRef] [Google Scholar]
  18. Kämpfer, P., Ruppel, S. & Remus, R.(2005).Enterobacter radicincitans sp. nov., a plant growth promoting species of the family Enterobacteriaceae. Syst Appl Microbiol 28, 213–221.[CrossRef] [Google Scholar]
  19. Kosako, Y., Tamura, K., Sakazaki, R. & Miki, K.(1996).Enterobacter kobei sp. nov., a new species of the family Enterobacteriaceae resembling Enterobacter cloacae. Curr Microbiol 33, 261–265.[CrossRef] [Google Scholar]
  20. Li, X., Zhang, D., Chen, F., Ma, J., Dong, Y. & Zhang, L.(2004).Klebsiella singaporensis sp. nov., a novel isomaltulose-producing bacterium. Int J Syst Evol Microbiol 54, 2131–2136.[CrossRef] [Google Scholar]
  21. Marmur, J.(1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.[CrossRef] [Google Scholar]
  22. Mollet, C., Drancourt, M. & Raoult, D.(1997).rpoB sequence analysis as a novel basis for bacterial identification. Mol Microbiol 26, 1005–1011.[CrossRef] [Google Scholar]
  23. Nicholas, K. B. & Nicholas, H. B., Jr (1997).GeneDoc: a tool for editing and annotating multiple sequence alignments. http://www.nrbsc.org/downloads/
  24. O'Hara, C. M., Steigerwalt, A. G., Hill, B. C., Farmer, J. J., III, Fanning, G. R. & Brenner, D. J.(1989).Enterobacter hormaechei, a new species of the family Enterobacteriaceae formerly known as enteric group 75. J Clin Microbiol 27, 2046–2049. [Google Scholar]
  25. Pavan, M. E., Franco, R. J., Rodriguez, J. M., Gadaleta, P., Abbott, S. L., Janda, J. M. & Zorzópulos, J.(2005). Phylogenetic relationships of the genus Kluyvera: transfer of Enterobacter intermedius Izard et al. 1980 to the genus Kluyvera as Kluyvera intermedia comb. nov. and reclassification of Kluyvera cochleae as a later synonym of K. intermedia. Int J Syst Evol Microbiol 55, 437–442.[CrossRef] [Google Scholar]
  26. Peng, G., Wang, H., Zhang, G., Hou, W., Liu, Y., Wang, E. T. & Tan, Z.(2006).Azospirillum melinis sp. nov., a group of diazotrophs isolated from tropical molasses grass. Int J Syst Evol Microbiol 56, 1263–1271.[CrossRef] [Google Scholar]
  27. Reinhold-Hurek, B., Hurek, T., Gillis, M., Hoste, B., Vancanneyt, M., Kersters, K. & De Ley, J.(1993).Azoarcus gen. nov., nitrogen-fixing proteobacteria associated with roots of Kallar grass (Leptochloa fusca (L.) Kunth), and description of two species, Azoarcus indigens sp. nov. and Azoarcus communis sp. nov. Int J Syst Bacteriol 43, 574–584.[CrossRef] [Google Scholar]
  28. Saitou, N. & Nei, M.(1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. [Google Scholar]
  29. Sasser, M.(1990).Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Newark, DE: Microbial ID, Inc.
  30. Sneath, P. H. A. & Sokal, R. R.(1973).Numerical Taxonomy. The Principles and Practice of Numerical Classification. San Francisco: W. H. Freeman.
  31. Stephan, R., Van Trappen, S., Cleenwerck, I., Vancanneyt, M., De Vos, P. & Lehner, A.(2007).Enterobacter turicensis sp. nov. and Enterobacter helveticus sp. nov., isolated from fruit powder. Int J Syst Evol Microbiol 57, 820–826.[CrossRef] [Google Scholar]
  32. Tan, Z. Y., Wang, E. T., Peng, G. X., Zhu, M. E., Martínez-Romero, E. & Chen, W. X.(1999). Characterization of bacteria isolated from wild legumes in the north-western regions of China. Int J Syst Bacteriol 49, 1457–1469.[CrossRef] [Google Scholar]
  33. Tan, Z., Hurek, T., Gyaneshwar, P., Ladha, J. K. & Reinhold-Hurek, B.(2001). Novel endophytes of rice form a taxonomically distinct subgroup of Serratia marcescens. Syst Appl Microbiol 24, 245–251.[CrossRef] [Google Scholar]
  34. Thompson, J. D., Higgins, D. G. & Gibson, T. J.(1994).clustalw: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[CrossRef] [Google Scholar]
  35. Tou, C. & Zhou, F.(1989). Non-nodular endorhizospheric nitrogen fixation in wetland rice. Can J Microbiol 35, 403–408.[CrossRef] [Google Scholar]
  36. Ueda, T., Suga, Y., Yahiro, N. & Matsuguchi, T.(1995). Remarkable N2-fixing bacterial diversity detected in rice roots by molecular evolutionary analysis of nifH gene sequences. J Bacteriol 177, 1414–1417. [Google Scholar]
  37. Van de Peer, Y. & De Wachter, R.(1994).treecon for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570. [Google Scholar]
  38. Vaughan, D. A.(1989). The genus Oryza L. Current status of taxonomy. Manila IRRI Res Pap Ser 138, 9 [Google Scholar]

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Transmission electron micrograph of a cell of strain Ola 51 showing a rod with the flagellar structure. Bar, 0.2 µm.


IS-PCR fingerprinting patterns of diazotrophic isolates of group A from the wild rice species and closely related species. M, Standard DNA marker; lanes 1–9, ATCC 13048 , ATCC 13047 , DSM 16656 and group A isolates Ola 51 , Ola 10, Ola 50, Ola 12, Ola 01 and Ola 28.


Whole-cell protein SDS-PAGE patterns of diazotrophic isolates of group A from and closely related reference strains. M, Molecular-mass standards; lanes 1–8, ATCC 13048 , ATCC 13047 and group A isolates Ola 51 , Ola 10, Ola 50, Ola 12, Ola 01 and Ola 28. Arrows indicate intense protein bands that differ between ATCC 13047 and the diazotrophic isolates.


Neighbour-joining dendrogram based on gene sequences of sp. nov. Ola 51 and closely related species. [PDF](30 KB)


[PDF file of Supplementary Tables S1-S3](98 KB)

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